Abstract:Volatile chemicals collected
from virgin Eurytoma amygdali females by extracting the
whole or parts of their bodies in methylene chloride were attractive
to males in laboratory bioassays. The same attraction was found
when volatile compounds were collected by the SMPE fibre. Comparative
GC-MS analysis of male and female extracts indicated differences
which corresponded to a number of peaks present only in the female
extract. Preparative GC fractionation of these peaks and subsequent
bioassays elicited male response. These peaks correspond to mono-unsaturated
and di-unsaturated long chain hydrocarbons as indicated by mass
spectrometric analysis.

Key words: Almond seed wasp, Eurytoma amygdali,
sex pheromone

Introduction

The almond seed wasp Eurytoma
amygdali Enderlein, (Hymenoptera:Eurytomidae) is considered
as one of the most important pests of almonds in a number of southeastern
countries, the Middle East and some of the countries of the former
Soviet Union. The damage caused to the crop can reach up to 90%
by mummifying the infested almond. Chemical control of this pest
is usually accomplished by the application of a systemic insecticide.
Since time of application is critical, monitoring of adult emergence
is carried out by placing cages containing infested almonds from
the previous year in the field and counting the emerging adults.
Pittara and Katsoyannos have reported male attraction to virgin
females in E. amygdali (Pittara and Katsoyannos 1985).
Katsoyannos et al. have used traps baited with virgin females
for monitoring the population of E. amygdali (Katsoyannos
et al. 1992). Due to the nature of this trap set-up
(living female wasps), its use is limited and can be applied only
from trained personnel. Since the chemical structure and composition
of the sex pheromone has not been elucidated yet, the use of synthetic
sex pheromone traps which could improve the monitoring process
is not yet possible. To the best of our knowledge there is no
reference in the literature so far for determination of sex pheromones
in Eurytomidae.

This work reports on the chemical
composition of cuticular extracts from male and female E. amygdali
and the potent chemicals which are involved in the mating process
of this wasp.

Material and methods

InsectsThe insects used
in this study were obtained from infested almonds collected from
the field and stored at 4 - 6 ° C in the dark. Adults were
obtained from almonds that had been transferred from the cold
room to the lab which was kept at 25 ± 2 ° C and a
14:10 hr light-dark regime. Adults were sex separated as soon
as they emerged and kept in cages under the conditions mentioned
above. Wasps were provided with a 10% sucrose solution.

Laboratory BioassaysBioassays were performed
in a 50 x 40 x 30 cm screen cage;
10-20, 1 to 5 days old males
were present in the cage during the bioassay. All bioassays were
conducted 3 to 4 hr after the onset of the photoperiod. A 2 x
2 cm Whatman No 1 filter paper impregnated with the extract to
be tested (2-3 female equivalent) was hung 5 cm below the top
of the cage and served as the source. A fan was used to generate
the air stream. Males exhibiting wing raising, lateral swing of
the body, attraction and movement towards the source and source
touching was the response criterion. The SPME fibre that had been
used to collect analytes from E. amygdali was also evaluated
for male response. The SPME holder was standing on the top of
the cage in such a place that allowed the exposed fibre to penetrate
ca. 5 cm inside the cage from the top part. The rest of the experimental
set-up was kept the same.

Solvent ExtractionCuticular compounds
were extracted with methylene chloride for 20
min from groups of male or female individuals using the whole
body or different parts of their bodies (head, thorax and abdomen).

Solid Phase Micro Extraction
(SPME)Collection of chemicals
from the insects cuticle was performed by rubbing a SPME fibre
(polydimethylsiloxane, SUPELCO, USA) onto the insect body. Two
individuals were used for each collection. The same SPME fibre
was also used to collect airborn volatiles by headspace extraction.
In this case, four individuals were placed in a 4ml vial and the
fibre was exposed to the volatiles for two hours.

Preparative Gas ChromatographyPreparative gas
chromatography was carried out on a Varian 3400 gas chromatograph
equipped with a FID detector and a 30 m x 0.53 mm x 1 µm
film thickness DB-5 column (J&W Scientific). The oven temperature
program was 50 °C for 2 min, then 5 °C/min to 280 °C
hold for 60 min. Helium was used as the carrier gas at a flow
rate of 3 ml/min. On column injections were made (1 µl)
at an injector temperature of 250 °C and a detector temperature
of 300 °C. The outlet of the column was split by a manually
operated metering valve between the detector (10%) and the collecting
capillary (90%). The fractions were collected in ice cooled fused
silica uncoated capillaries and were subsequently washed with
a few µls of methylene chloride.

Gas Chromatography - Mass
SpectrometryGas chromatography-mass
spectrometry analysis was carried out on a Hewlett Packard 5890
Series II gas chromatograph interfaced to a Fisons VG Trio 1000
(Manchester M23 9BE, UK) quadrupole mass spectrometer. Electron
impact ionisation was used, with an electron energy of 70 eV and
a trap current of 200 µA. All extracts were chromatographed
on a 60 m x 0.25 mm x 0.1 µm film thickness DB-5 column
(J&W Scientific). The oven temperature program was 50 °C
for 2 min, then 5 °C/min to 250 °C hold for 1 min, then
2 °C/min to 280 °C and hold for 50 min. Helium was used
as the carrier gas at a flow rate of 1 ml/min. Splitless injections
were made (1 µl) at an injector temperature of 250 °C
and a splitless period of 90 s. For the SPME analysis, the fibre
was desorbed into the injection port for 5 min.

Results and discussionExtracts of E. amygdali
females from all three techniques evaluated elicited male response
when tested in laboratory bioassays. On the contrary male response
to male whole-body extract was not significantly different from
the control. In order to identify the female body part(s) involved
in the release of those chemicals responsible for the mating process
of E. amygdali, male attraction to extracts of head, thorax
and abdomen was evaluated in the lab. It was found that thorax
extracts elicited significantly greater response than head and
abdomen extracts (Fig. 1). This is in accordance with the work
described recently by Leal et al. for Bephratelloides
pomorum (Hymenoptera, Eurotomidae) (Leal et al. 1997).

Comparative GC-MS analysis of
female extracts with all three techniques tested, exhibited a
similar chromatographic profile with mainly quantitative rather
than qualitative differences. The same applies to male extracts.
The results indicate that the SPME technique compares well with
the conventional solvent extraction approach and offers a fast,
solventless alternative to it. Differences were observed though,
between male and female extracts regardless the technique used.
The vast majority of compounds identified are long chain saturated,
mono-unsaturated and di-unsaturated hydrocarbons. The saturated
hydrocarbon chromatographic profile from each sex was qualitatively
similar with some quantitative differences. Most of the alkenes
and both alkadienes identified were present only in the female
extract (Fig. 2). It has been reported that for Cardiochiles
nigriceps (Hymenoptera: Braconidae) alkadienes which are present
only in the female extract were responsible for mediating courtship
behaviour (Syvertsen et al., 1995). For this species, the alkanes
fraction from each sex were qualitatively simiral and to a lesser
extent the same was applied for the alkenes fraction. Further
fractionation of the crude female extract on preparative GC led
to the collection of three distinct fractions containing compounds
that had been detected only in the female extract. Fraction 1
contained tricosene and tricosadiene, fraction 2 contained pentacosene
and pentacosadiene and fraction 3 contained triaconteneand
a coeluting saturated hydrocarbon (Fig. 2). Bioassays performed
with these fractions revealed that the highest male response was
elicited by fraction 1 and the lowest by fraction 3. The sex pheromones
isolated from some parasitic Hymenoptera have been determined
to consist of multiple compounds (Shu and Jones 1993; Swedenborg
and Jones 1992; Swedenborg et al. 1993). Our results suggest that
alkadienes and to a lesser extent alkenes identified in the female
extract of E. amygdali are potent chemicals responsible
for the mating process of this wasp.

The chemical composition of
male and female E. amygdali cuticular extracts has been
determined. The major differences between the chemical composition
of the two sexes are alkenes and alkadienes which are present
only in the female extract. The results presented in this work
suggest that mono-unsaturated and di-unsaturated long chain hydrocarbons
elicit male response. Further work will include complete elucidation
of the structure of these compounds and bioassays to unambiguously
determine the compound(s) which are responsible for the mating
process of the almond seed wasp.